Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Specific suppression of the host""s immune response to donor HLA antigens remains the ultimate goal for clinical transplantation. In spite of considerable effort, however, allospecific human suppressor T cells (Ts) have been difficult to generate. The studies herein (first series of experiments) show that allospecific and xenospecific Ts can be raised by multiple priming of human T cells in mixed lymphocyte cultures (MLC). Ts derive from the CD8+CD28xe2x88x92 subset and recognize specifically the MHC class I antigens expressed by Antigen-Presenting Cells (APC) used for in vitro immunization. Allospecific Ts prevent the upregulation of B7 molecules on target APCs, interfering with the CD28-B7 interaction required for T helper (Th) activation. These findings provide a basis for the development of specific immunosuppressive therapy.
The induction of donor-specific tolerance remains the ultimate goal for clinical transplantation. Immunosuppressive treatments that have been developed so far act non-specifically, placing the recipient at increased risk for infections and malignancies.
Transplant tolerance has been induced in adult animals by inactivation or depletion of mature T lymphocytes prior to transplantation using cyclosporine (CsA) (1), total lymphoid irradiation (2,3), anti-lymphocyte serum (4), antibodies against CD4+ and CD8+ T cells (5), or donor-specific transfusions (6,7). Studies of peripheral graft tolerance have suggested the existence of an active mechanism of suppression which is donor-specific and can be transferred adoptively to secondary hosts (1, 7-10). However, there is still controversy concerning the phenotypic characteristics of these regulatory T cells and their MHC restriction, as both CD8+ and CD4+ T cells were reported to display suppressive activity (11). This controversy lead to the speculation that no distinctive Ts lineage actually exists. It has been suggested that suppression may result from antagonistic effects of (Th)2-type lymphokines (such as IL-4 and IL-10) on the response of Th1 cells (2,12), or from recognition by Ts of either idiotypic determinants of the TCR of alloreactive T cells or of MHC antigens expressed on stimulating cells (10,13). The generation of Ts lines has proven, however, to be a difficult task rendering the characterization of these cells hard to achieve.
The aim of the present study (first series of experiments) was to develop and characterize suppressor T cell lines which inhibit specifically the alloimmune response. This study established for the first time the existence of a population of CD8+CD28xe2x88x92 Ts which are allorestricted by HLA-class I antigens expressed by the cells used for priming. The mechanism of suppression is based on the capacity of Ts to prevent the upregulation of B7 molecules (CD80 and CD86) induced by Th on the stimulating APC. Allorestricted Ts can be easily and reproducibly expanded in cultures facilitating the in vitro study of immunoregulatory networks and the development of new strategies for specific immunosuppression.
Evidence that T cells can down-regulate the immune response by producing or consuming certain cytokines or by lysing APCs or T helper cells has been provided in various systems. However, the generation and characterization of suppressor T cell lines have met with limited success. In the second series of experiments herein it is shown that xenospecific suppressor T cells can be generated by in vitro stimulation of human T cells with pig APCs. Similar to allospecific suppressors, these xenospecific suppressor T cells carry the CD8+CD28xe2x88x92 phenotype and react to MHC class I antigens expressed by the APCs used for priming. TCR spectratyping of T suppressor cells showed oligoclonal usage of TCR-Vxcex2 families, indicating that xenostimulation of CD8+CD28xe2x88x92 T cells results in antigen-driven selection of a limited Vxcex2 repertoire. Xenospecific T suppressor cells prevent the up-regulation of CD154 molecules on the membrane of T helper (Th) cells, inhibiting their ability to react against the immunizing MHC-class II xenoantigens. The mechanism of this suppression, therefore, appears to be blockade of CD154/CD40 interaction required for efficient costimulation of activated T cells.
The induction of regulatory T cells may offer an effective means for specific immunosuppression of autoimmune disease and allograft rejection. The existence of suppressor T cells has been previously documented, yet their mechanism of action remains poorly characterized. The third series of studies herein demonstrate that T suppressor (Ts) cell lines can be generated by in vitro immunization of human PBMCs, with synthetic peptides or soluble proteins coupled to beads. Such Ts cells express the CD8+CD28xe2x88x92 phenotype and show the following characteristics: a) antigen specificity and restriction by self MHC Class I molecules, b) limited TCR V beta gene usage, c) ability to inhibit antigen-specific, MHC Class II restricted, Th proliferative responses, and d) capacity to downregulate and/or inhibit the upregulation by Th of CD40, CD80, and CD86 molecules on APCs. The inhibitory activity of Ts on Th proliferation requires the tripartite interaction between Th, Ts, and APCs and results from inefficient costimulation of Th.
Understanding the mechanism which underlies the induction of immunologic tolerance is crucial to the development of strategies for treatment of auto-immune diseases and allograft rejection. Although the concept that T suppressor cells (Ts) downregulate the immune response has long been accepted, the existence of a distinct population of lymphocytes that mediates suppression has not been convincingly demonstrated. In previous studies, human T cell lines (TCLs) were utilized to analyze the suppressive effects of CD8+ CD28xe2x88x92 T cells in allogeneic, peptide specific and xeno-specific responses. In each case, CD8+ CD26xe2x88x92 T cells inhibit proliferation of CD4+ T helper lymphocytes (Th) with cognate antigen specificity. These CD8+ CD28xe2x88x92 T cells display the critical functional characteristics of T suppressor cells. Similar to the induction of CD8+ cytotoxic T cells (Tc) by Th, this process depends on antigen presenting cells (APC) acting as a xe2x80x9cbridgexe2x80x9d between MHC-class I specific CD8 and class II specific CD4+ T cells. A possible explanation of Ts-mediated suppression is their ability to modulate the function of APCs. The fourth series of studies herein show that CD8+ CD28xe2x88x92 Ts directly inhibit the CD40 signaling pathway of APC by a contact-dependent mechanism that renders bridging APCs incapable of inducing CD4+ Th activation. The effects of Ts on the functional state of APC supports the concept that the order in which Ts and Th cells interact with cognate APCs determines the functional outcome of immune responses.
This invention provides a method of generating antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells which comprises: a) obtaining peripheral blood T cells from a subject; b) stimulating by multiple priming a T cell line from the T cells obtained in step (a) with allogeneic antigen presenting cells (APCs), said APCs expressing an MHC class I antigen recognized by the primed T cell line and an MHC class II antigen recognized by CD4+ T helper cells from said primed T cell line; c) isolating primed CD8+ T cells and CD4+ T helper cells from the T cell line stimulated in step (b); d) isolating primed CD8+CD28xe2x88x92 T cells from the isolated primed CD8+ T cells of step (c); e) detecting suppression by the primed CD8+CD28xe2x88x92 T cells isolated in step (d) of interaction between the CD4+ T helper cells isolated in step (c) and allogeneic antigen presenting cells (APCs) expressing the same MHC class I antigen and the same MHC class II antigen expressed by the APCs used to stimulate the T cell line of step (b), thereby identifying antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells; and f) expanding the antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells identified in step (e), thereby generating the antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells.
This invention provides antigen specific allospecific human suppressor CD8+CD28+ T cells produced by the method of generating antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells which comprises: a) obtaining peripheral blood T cells from a subject; b) stimulating by multiple priming a T cell line from the T cells obtained in step (a) with allogeneic antigen presenting cells (APCs), said APCs expressing an MHC class I antigen recognized by the primed T cell line and an MHC class II antigen recognized by CD4+ T helper cells from said primed T cell line; c) isolating primed CD8+ T cells and CD4+ T helper cells from the T cell line stimulated in step (b); d) isolating primed CD8+CD28xe2x88x92 T cells from the isolated primed CD8+ T cells of step (c); e) detecting suppression by the primed CD8+CD28xe2x88x92 T cells isolated in step (d) of interaction between the CD4+ T helper cells isolated in step (c) and allogeneic antigen presenting cells (APCs) expressing the same MHC class I antigen and the same MHC class II antigen expressed by the APCs used to stimulate the T cell line of step (b), thereby identifying antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells; and f) expanding the antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells identified in step (e).
This invention provides a method of generating xenospecific human suppressor CD8+CD28xe2x88x92 T cells which comprises: a) obtaining peripheral blood T cells from a human subject; b) stimulating by multiple priming a human T cell line from the T cells obtained in step (a) with xenogeneic mammalian antigen presenting cells (APCs), said APCs expressing a xenogeneic MHC class I antigen and a xenogeneic MHC class II antigen; c) isolating primed human CD8+ T cells and human CD4+ T helper cells from the T cell line stimulated in step (b); d) isolating primed human CD8+CD28xe2x88x92 T cells from the isolated primed human CD8+ T cells of step (c); e) detecting suppression by the primed human CD8+CD28xe2x88x92 T cells isolated in step (d) of interaction between the human CD4+ T helper cells isolated in step (c) and xenogeneic antigen presenting cells (APCs) expressing the same xenogeneic MHC class I antigen and xenogeneic MHC class II antigen expressed by the xenogeneic APCs used to stimulate the human T cell line of step (b), thereby identifying xenospecific human suppressor CD8+CD28xe2x88x92 T cells; f) expanding the xenospecific human suppressor CD8+CD28xe2x88x92 T cells identified in step (e), thereby generating the xenospecific human suppressor CD8+CD28xe2x88x92 T cells.
This invention provides xenospecific human suppressor CD8+ CD28+ T cells produced by the method of generating xenospecific human suppressor CD8+CD28xe2x88x92 T cells which comprises: a) obtaining peripheral blood T cells from a human subject; b) stimulating by multiple priming a human T cell line from the T cells obtained in step (a) with xenogeneic mammalian antigen presenting cells (APCs), said APCs expressing a xenogeneic MHC class I antigen and a xenogeneic MHC class II antigen; c) isolating primed human CD8+ T cells and human CD4+ T helper cells from the T cell line stimulated in step (b); d) isolating primed human CD8+CD28xe2x88x92 T cells from the isolated primed human CD8+ T cells of step (c); e) detecting suppression by the primed human CD8+CD28xe2x88x92 T cells isolated in step (d) of interaction between the human CD4+ T helper cells isolated in step (c) and xenogeneic antigen presenting cells (APCS) expressing the same xenogeneic MHC class I antigen and xenogeneic MHC class II antigen expressed by the xenogeneic APCs used to stimulate the human T cell line of step (b), thereby identifying xenospecific human suppressor CD8+CD28xe2x88x92 T cells; and f) expanding the xenospecific human suppressor CD8+CD28xe2x88x92 T cells identified in step (e).
This invention provides a method of generating allopeptide antigen specific human suppressor CD8+CD28xe2x88x92 T cells which comprises: a) obtaining peripheral blood T cells from a subject; b) stimulating by multiple priming a T cell line from the T cells obtained in step (a) with autologous antigen presenting cells (APCs) pulsed with an allopeptide, said allopeptide comprising an amino acid sequence comprising both MHC class I and MHC class II amino acid sequences wherein the amino acid sequences are binding sequences (motifs) and are recognized by the primed T cell line; c) isolating primed CD8+ T cells and CD4+ T helper cells from the T cell line stimulated in step (b); d) isolating primed CD8+CD28xe2x88x92 T cells from the isolated primed CD8+ T cells of step (c); e) detecting suppression by the primed CD8+CD28xe2x88x92 T cells isolated in step (d) of interaction between the CD4+ T helper cells isolated in step (c) and autologous antigen presenting cells (APCs) expressing the same MHC class I and MHC class II binding motifs as expressed by the APCs used to stimulate the T cell line of step (b), thereby identifying allopeptide antigen specific human suppressor CD8+CD28xe2x88x92 T cells; and f) expanding the allopeptide antigen specific human suppressor CD8+CD28xe2x88x92 T cells identified in step (e), thereby generating the antigen specific human suppressor CD8+CD28xe2x88x92 T cells.
This invention provides an antigen specific human suppressor CD8+CD28xe2x88x92 T cells produced by the above-described method of generating the antigen specific human suppressor CD8+CD28xe2x88x92 T cells.
This invention provides a method of determining whether a level of immunosuppresant therapy given to a subject undergoing the level immunosuppression therapy requires a reduction which comprises: a) obtaining a blood sample from the subject; and b) determining the presence of T suppressor cells present in the sample, the presence of T suppressor cells indicating that the subject requires the reduction of immunosuppresant therapy.
This invention provides a method of reducing the risk of rejection of an allograft in a subject undergoing immunosuppression therapy which comprises: a) obtaining a blood sample from the subject; b) removing T suppressor cells from the blood sample; c) expanding the T suppressor cells of step (b); and d) reintroducing the expanded T suppressor cells of step (b) into the subject.
This invention provides a method of reducing the level of rejection of an allograft in a subject undergoing immunosuppression therapy which comprises administering to the subject the T suppressor cells produced by the above-described method of generating antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells, thereby preventing rejection of the tissue or organ transplant in the subject.
This invention provides a method of reducing the level of rejection of an allograft in a subject undergoing immunosuppression therapy which comprises administering to the subject the T suppressor cells produced by the above-described method of generating allopeptide antigen specific human suppressor CD8+CD28xe2x88x92 T cells, thereby preventing rejection of the tissue or organ transplant in the subject.
This invention provides a method of preventing rejection of an allograft in a subject which comprises: a) obtaining a blood sample from the subject; b) removing T suppressor cells from the blood sample; c) expanding the T suppressor cells of step (b); and d) reintroducing the expanded T suppressor cells of step (b) into the subject, thereby preventing the rejection of the allograft in the subject.
This invention provides a method of preventing rejection of an allograft in a subject which comprises administering the T suppressor cells produced by the above-described method of generating antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells to the subject, thereby preventing rejection of the allograft in the subject.
This invention provides a method of preventing rejection of an allograft in a subject which comprises administering the T suppressor cells produced by the above-described method of generating allopeptide antigen specific human suppressor CD8+CD28xe2x88x92 T cells to the subject, thereby preventing rejection of the allograft in the subject.
This invention provides a method of preventing rejection of a xenograft in a subject which comprises: a) obtaining a blood sample from the subject; b) removing T suppressor cells from the blood sample; c) expanding the T suppressor cells of step (b); and d) reintroducing the expanded T suppressor cells of step (b) into the subject, thereby preventing the rejection of the xenograft in the subject.
This invention provides a method of preventing rejection of a xenograft in a subject which comprises administering the T suppressor cells produced by the above-described method of generating xenospecific human suppressor CD8+CD28xe2x88x92 T cells to the subject, thereby preventing rejection of the xenograft in the subject.
This invention provides a method of preventing autoimmune disease in a subject which comprises: a) obtaining a blood sample from the subject; b) removing T suppressor cells from the blood sample; c) expanding the T suppressor cells of step (b); and d) reintroducing the expanded T suppressor cells of step (b) into the subject, thereby preventing autoimmune disease in the subject.
This invention provides a method of preventing autoimmune disease in a subject which comprises administering the T suppressor cells produced by the above-described method of generating antigen specific allospecific human suppressor CD8+CD28xe2x88x92 T cells to the subject, thereby preventing autoimmune disease in the subject.
This invention provides a method of preventing autoimmune disease in a subject which comprises administering the T suppressor cells produced by above-described method of generating allopeptide antigen specific human suppressor CD8+CD28xe2x88x92 T cells to the subject, thereby preventing autoimmune disease in the subject.
This invention provides a vaccine comprising allospecific T suppressor cells stimulated by APCs expressing an MHC class I antigen and an MHC class II antigen which T suppressor cells suppress an interaction between CD4+ T helper cells and allogeneic antigen presenting cells (APCs) expressing the same MHC class I antigen and the same MHC class II antigen expressed by the APCs used to stimulate the allospecific T suppressor cells.
This invention provides a vaccine comprising xenospecific T suppressor cells stimulated by APCs expressing a xenogeneic MHC class I antigen and a xenogeneic MHC class II antigen which xenospecific T suppressor cells suppress an interaction between CD4+ T helper cells and xenogeneic antigen presenting cells (APCs) expressing the same xenogeneic MHC class I antigen and xenogeneic MHC class II antigen expressed by the APCs used to stimulate the xenospecific T suppressor cells.
This invention provides a method of inducing anergic T helper cells which comprises: a)incubating antigen presenting cells (APC) with allospecific T suppressor cells (Ts); b) overexpressing in the APC mRNA which encodes at least one monocyte inhibitory receptor (MIR), in a mixture of cells comprising the APCs from step (a), wherein overexpression of MIR transmits negative inhibitory signals to recruit an inhibitory signaling molecule, thyrosine phosphatase SHP-1 such that the APC are rendered tolerogenic; and c) incubating the APCs from step (b) with T helper cells (Th) to induce Th anergy.
This invention provides a method of generating a tolerogenic antigen presenting cell (APC) which comprises: a) contacting the APC with Ts; and b) overexpressing mRNA which encodes an MIR in the APC, thereby generating a tolerogenic antigen presenting cell (APC).
This invention provides a method of reducing the level of rejection of an allograft tissue or organ in a subject who is a transplant recipient of the allograft tissue or organ which comprises administering to the subject tolerogenic antigen presenting cells (APC) which overexpress monocyte inhibitory receptor (MIR), wherein the APC have been incubated with Ts prior to overexpression of MIR, thereby inducing Th anergy so as to prevent rejection of the tissue or organ allograft in the subject.
This invention provides a method of suppressing an autoimmune disease in a subject which comprises: a) contacting antigen presenting cells (APC) of the subject with T suppressor cells (Ts) specific for the antigen which induces the autoimmune disease; and b) administering to the subject the APC of step(a), thereby inducing tolerance to the antigen so as to suppress the autoimmune disease in the subject.
This invention provides a method of suppressing an autoimmune disease in a subject which comprises: a) overexpressing monocyte inhibitory receptor (MIR) in antigen presenting cells (APC) of the subject, which APC present the antigen which induces the autoimmune disease; and b) administering to the subject the APC of step (a), thereby inducing tolerance to the antigen so as to suppress the autoimmune disease in the subject.
This invention provides a method of inducing tolerance to an allograft tissue or organ in a subject which comprises administering to the subject tolerogenic antigen presenting cells (APC) which overexpress monocyte inhibitory receptor (MIR), thereby inducing tolerance to the allograft in the subject.
This invention provides a method of inducing tolerance to a xenograft tissue or organ in a subject which comprises administering to the subject tolerogenic antigen presenting cells (APC) which overexpress monocyte inhibitory receptor (MIR), thereby inducing tolerance to the xenograft in the subject.
This invention provides an antigen presenting cell (APC) which overexpresses ILT3, wherein the APC comprises a retroviral vector comprising a nucleic acid sequence which encodes ILT3 and ILT4 and overexpresses ILT3 and ILT4.
This invention provides a method of inducing tolerance to a xenograft tissue or organ transplant in a subject which comprises: a) introducing to an antigen presenting cell (APC) of a tissue or organ transplant donor a vector which overexpresses ILT3, wherein the vector comprises a nucleic acid sequence which encodes ILT3 and overexpresses ILT3; and b) administering the APC of step (a) to the subject, thereby inducing tolerance to the xenograft in the subject.
This invention provides a method of inducing tolerance to an allograft tissue or organ in a subject which comprises: a) introducing to an antigen presenting cell (APC) of a tissue or organ transplant donor a vector which overexpresses ILT3, wherein the vector comprises a nucleic acid sequence which encodes ILT3 and overexpresses ILT3; and b) administering the APC of step (a) to the subject, thereby inducing tolerance to the allograft in the subject.
This invention provides a method of treating an autoimmune disease in a subject which comprises: a) introducing to an antigen presenting cell (APC) of a subject having the autoimmune disease a vector which overexpresses ILT3, wherein the vector comprises a nucleic acid sequence which encodes ILT3 and overexpresses ILT3; and b) administering the APC of step (a) to the subject, thereby treating the autoimmune disease in the subject.
This invention provides a method of determining the appearance of T suppressor (Ts) cells which comprises detecting the level of expression of ILT3, ILT4 and ILT2 protein in APCs of a subject, wherein the subject is a xenograft tissue or organ transplant recipient which comprises: a) obtaining a sample from the subject; and b) detecting in the sample of step (a) overexpression of mRNA which encodes the ILT3, ILT4 and ILT2 protein in the APC of the subject, wherein detection of overexpression of mRNA which encodes the ILT3, ILT4 and ILT2 protein indicates the appearance of T suppressor cells in the subject.
This invention provides a method of determining the appearance of T suppressor (Ts) cells which comprises detecting the level of expression of ILT3, ILT4 and ILT2 protein in APCs of a subject, wherein the subject is an allograft tissue or organ transplant recipient which comprises: a) obtaining a sample from the subject; and b) detecting in the sample of step (a) overexpression of mRNA which encodes the ILT3, ILT4 and ILT2 protein in the APC of the subject, wherein detection of overexpression of mRNA which encodes the ILT3, ILT4 and ILT2 protein indicates the appearance of T suppressor cells in the subject.